The present invention relates to the field of molecular genetics, including the identification and detection of certain nucleotide sequences.
The nucleic acids comprising the genome of an organism contain the genetic information for that organism. The translation or expression of these nucleic acids generates proteins that function in many diverse ways within the organism. Even minute changes in a nucleotide sequence, including single base pair substitutions, can have a siginificant effect in the quality or quantity of a protein. Single nucleotide changes are referred to as single nucleotide polymorphisms or simply SNPs, and the site at which the SNP occurs is typically referred to as a polymorphic site.
Many SNPs, as well as larger nucleic acid alterations, can affect the phenotype of the organism, and in some instances can result in the onset of disease. For example, diseases associated with SNPs include: sickle cell anemia, xcex2-thalassemias, diabetes, cystic fibrosis, hyperlipoproteinemia, a wide variety of autoimmune diseases, and the formation of oncogenes. In addition to causing or affecting disease states, point mutations can cause altered pathogenicity and resistance to therapeutics that target certain microorganisms.
The ability to detect specific nucleotide alterations or mutations in DNA sequences has a number of medical and non-medical utilities. For example, methods capable of identifying nucleotide alterations provide a means for screening and diagnosing many common diseases that are associated with SNPs. Methods that can quickly identify such changes or mutations are also valuable in taking prophylactic measures, assessing the propensity for disease, and in patient counseling and education. As for non-medical applications, such methods have value in the detection of microorganisms, resolving paternity disputes and in forensic analysis to identify perpetrators of crimes.
Various methods have been developed to obtain sequence information for variant sites. Such methods include hybridization reactions between a target nucleic acid and allele-specific oligonucleotidc (ASO) probes (see, e.g., European Patent Publications EP-237362 and EP-32931 1), allele specific amplification (see, e.g., U.S. Pat. Nos. 5,521,301; 5,639,611; and 5,981,176), mini-sequencing methods, quantitative RT-PCR methods (eg., the so-called xe2x80x9cTaqMan assaysxe2x80x9d; see, e.g., U.S. Pat. No. 5,210,015 to Gelfand, U.S. Pat. No. 5,538,848 to Livak, et al., and U.S. Pat. No. 5,863,736 to Haaland, as well as Heid, C.A., et al. Genome Research, 6:986-994 (1996); Gibson, U .E. M, et al., Genome Research 6:995-1001 (1996); Holland, P. M., et al. Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, K. J., et al., PCR Methods and Applications 357-362 (1995)), and various single base pair extension (SBPE) assays and related extension assays.
Extension assays typically involve hybridizing a primer that is complementary to a target nucleic acid such that the 3xe2x80x2 end of the primer is immediately 5xe2x80x2 of the variant site or is adjacent thereto. Extension is conducted in the presence of one or more labeled non-extendible nucleotides that are complementary to the nucleotide(s) that occupy the variant site and a polymerase. The non-extendible nucleotide is a nucleotide analog that prevents further extension by the polymerase once incorporated into the primer. If the added non-extendible nucleotide(s) is(are) complementary to the nucleotide at the variant site, then a labeled non-extendible nucleotide is incorporated onto the 3xe2x80x2 end of the primer to generate a labeled extension product. Hence, extended primers provide an indication of which nucleotide is present at the variant site of a target nucleic acid. Such methods are discussed, for example, in U.S. Pat. Nos. 5,846,710; 6,004,744; 5,888,819; 5,856,092; 5,710,028; and 6,013,431; and in PCT publication WO 92/16657, each of which is incorporated by reference.
Certain SBPE extension reactions suffer from various shortcomings. Some methods generate labeled extension product for only one of the two or more allelic forms of a target nucleic acid. This can be problematic as one cannot distinguish with certainty a failed experiment from the situation in which a sample does not contain a particular allelic form of a target nucleic acid. Often, the extension methods also generate extension products of the same size for different allelic forms of a target nucleic acid. Consequently, different alleles are frequently only distinguished based upon differences in how extension products generated from different allelic forms of the target nucleic acid are labeled (e.g., different extension products bear different labels, or one extension product is labeled whereas another extension product is unlabeled). This means that different allelic forms can be distinguished only by a single criterion.
The present invention provides methods and kits for detecting and identifying the nucleotide present at the variant site of a target nucleic acid of interest. The methods and kits can be utilized in research, clinical and laboratory settings. In general, the methods involve conducting extension reactions in the presence of a mixture of labeled extendible and labeled non-extendible nucleotides. The detection of incorporation of labeled nucleotide provides an indication of the identity of the nucleotide at the variant site since the incorporated nucleotide is complementary to the nucleotide at the site of variation. The methods can be used in conducting genotyping analyses and can be performed in multiplexing fonnats. The methods and kits have utility in diverse applications including, for example, analyzing point mutations and single nucleotide polymorphisms, detection of pathogens, paternity disputes, prenatal testing and forensic investigations.
Certain methods of the invention are methods of analyzing a variant site of a target nucleic acid and involve conducting a template-dependent extension reaction comprising extending a primer in the presence of the target nucleic acid and a mixture of labeled nucleotides comprising at least one labeled extendible nucleotide and at least one labeled non-extendible nucleotide, each complementary to a different allelic form of the target nucleic acid. The primer hybridizes to a segment of the target nucleic acid such that the 3xe2x80x2-end of the primer hybridizes adjacent the variant site of the target nucleic acid. If the labeled extendible nucleotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of the labeled extendible nucleotide. Whereas, if the labeled non-extendible nucleotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of the labeled non-extendible nucleotide. Incorporation of labeled nucleotide into the extended primer is then detected, with the identity of the labeled nucleotide incorporated into the primer indicating the identity of the nucleotide at the variant site. In some instances, the variant site is a bi-allelic site and the mixture contains a single labeled extendible nucleotide and a single labeled non-extendible nucleotide such as a dideoxynucleotide or an arabinoside triphosphate.
The invention further provides multiplexing methods in which multiple variant sites are analyzed at the same time. Certain of these methods involve conducting a plurality of template-dependent extension reactions in the presence of a plurality of different primers, wherein different primers hybridize adjacent to different variant sites of one or more target nucleic acids and are differentially labeled. Each extension reaction comprises contacting a sample containing the target nucleic acids with multiple copies of one of the different primers, wherein the primer bears a label and the 3xe2x80x2-end of the primer hybridizes adjacent to but not including the variant site of one of the target nucleic acids. The copies of the primer are then exposed to a mixture of nucleotides comprising at least one labeled extendible nucleotide and at least one labeled non-extendible nucleotide under conditions whereby if the labeled extendible nucleotide is complementary to the nucleotide occupying the variant site, at least one copy of the primer is extended by incorporation of labeled extendible nucleotide, whereas if the labeled non-extendible nucleotide is complementary to the nucleotide occupying the variant site, at least one copy of the primer is extended by incorporation of labeled non-extendible nucleotide. Collectively, the extension reactions generate a plurality of different extension products, extension products generated from different variant sites being distinguishable on the basis of the different labels borne by the extended primers. The incorporation of labeled nucleotides into the extension products is detected as an indication of the nucleotides occupying the site of variation in the target nucleic acids.
Kits for conducting certain methods of the invention are also provided by the invention. Some kits include a mixture of nucleotides comprising at least one labeled extendible nucleotide and at least one labeled non-extendible nucleotide, the nucleotides complementary to different allelic forms of a target nucleic acid. Also included is at least one primer, each primer hybridizing to a segment of the target nucleic acid such that the 3xe2x80x2 end of the primer is adjacent the variant site of the target nucleic acid. In some instances the primers and labeled nucleotides are selected for use with a SNP that is correlated with a particular disease.